Functional expression of the Flp recombinase in Mycobacterium bovis BCG

Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants because there are only two efficient antibiotic resistance genes available for allelic exchange experiments. Sequence-specific recombinbases such as the Flp recombinase can be used to excise resistance markers. Expression of the flp(e) gene from Saccharomyces cerevisiae is functional for this purpose in fast-growing Mycobacterium smegmatis but not in slow-growing mycobacteria such as M. bovis BCG or M. tuberculosis. We synthesized the flp(m) gene by adapting the codon usage to that preferred by M. tuberculosis. This increased the G+C content from 38% to 61%. Using the synthetic flp(m) gene, the frequency of removal of FRT-hyg-FRT cassette from the chromosome by the Flp recombinase was increased by more than 100-fold in M. smegmatis. In addition, 40% of all clones of M. bovis BCG had lost the hyg resistance cassette after transient expression of the flp(m) gene. Sequencing of the chromosomal DNA showed that excision of the FRT-hyg-FRT cassette by Flp was specific. These results show that the flp(m) encoded Flp recombinase is not only an improved genetic tool for M. smegmatis, but can also be used in slow growing mycobacteria such as M. tuberculosis for constructing unmarked mutations. Other more sophisticated applications in mycobacterial genetics would also profit from the improved Flp/FRT system.     

红酵母NZ-01发酵条件的优化

以红酵母菌株NZ-01为试验菌株,研究其发酵工艺与中试生产。采用摇瓶发酵优化的方式,研究培养基组分与发酵工艺条件对该菌发酵的影响,并进行中试放大生产。结果显示,该菌最适生长培养基组分为葡萄糖10g/L,蔗糖10g/L,酵母膏10g/L,牛肉膏2.5g/L;色素合成最适培养基组分为葡萄糖15g/L,蔗糖10g/L,酵母膏2.5g/L,牛肉膏5g/L。最适生长起始pH值为6.0,最适接种量为8%,生长周期为44h;最适色素合成起始pH值为7.0,最适色素合成接种量为8%,色素合成周期为48h。发酵优化后的色素产量3.88μg/mL较优化前1.71μg/mL提高了127%。中试产量达3.05μg/mL。红酵母菌NZ-01优化后的发酵条件可以应用于中试生产虾青素,有规模化生产应用潜力。     

重组人尿型纤溶酶原激活剂的中试生产与性质研究

表达uPA的CHOdhfr工程细胞CL11G在30升生物反应器中高密度大规模培养65天后,获得1200余升细胞培养上清;经阳离子交换层析、凝胶过滤、苯甲眯亲和层析和阴离子交换层析四步纯化工艺,获得uPA冻干纯品40克。对产品进行性质研究和质量检定,各项指标符合理论数据和《中国生物制品规程》。     

地鼠繁殖方式的改进与规模化生产

目的：探索SPF地鼠在屏障系统内的配种繁殖方式是否适合目前地鼠大规模生产,满足乙脑减毒活疫苗生产所需动物。方法：采取短期同居配种的繁殖方式进行试验和大规模生产。结果：短期同居配种的繁殖方式与人工刺激配种的繁殖方式相比具有仔鼠成活率高、受孕率高、单产高、利用率高的特点。结论：地鼠短期同居配种的繁殖方式适合在屏障系统内操作和大规模生产。     

Functional Equivalence of the Two Sides of the Human Nose in Odor Detection

Repeated measurements of detection thresholds for amyl acetateand acetic acid on different days demonstrated that the rightand left sides of the human nose are functionally equivalentin detecting odors. Chem. Senses 20: 585–587, 1995.     

区域尺度的中国植物功能型与生物群区

利用“生态－外貌”原则,中国的现状植被类型及其分布,确定中国的39种优势植物功能型：高山常绿针叶、北方常绿针叶、北方夏绿针叶、冷温带常绿针叶、温带常绿针叶、暖温带常绿阔叶、暖温带硬叶阔叶、暖温带夏绿阔叶、热带常绿阔叶、热带 雨绿阔叶、热带落叶阔叶、暖温带竹、高山／亚高山灌木、温带草原灌木、温带荒漠灌木、冷温带灌木、温带灌木、暖温带灌木、热带灌木、干旱灌木、高山草、荒漠草、温带草原草、温带草、沼泽草、红树、北方农作物、冷温带农作物、温带农作物、暖温带农作物、热带农作物和裸地。再依据优势植物功能型归并中国的21类潜在生物群区：北方（寒温带）落叶林、北方（寒温带）常绿林、冷温带针阔叶混交林、温带落叶阔叶林、暖温带（亚热带）落叶常绿阔叶混交林、暖温带（亚热带）常绿阔叶林、暖温带（亚）常绿阔叶季风林、热带雨林、热带季雨林、热带落叶林、红树林、干旱疏林／稀树草原、;温带草甸／稀树草原、温带草原、温带半草原、温带荒漠、温带半荒漠、高山／高山针叶林、高山／亚高山灌丛／草甸、高山／亚高山草原和高山／亚高山荒漠。如果考虑现状农业植被类型：一年一熟农作物、二年三熟农作物、一年二熟农作物和一年三熟农作物,可归并为25类现状生物群区。这是全球生态学和古生态学研究中区域尺度旧我国植物功能型和生物群区分类的一次尝试。     

Pilot Scale Bioremediation of Creosote-Contaminated Soil—Efficacy of Enhanced Natural Attenuation and Bioaugmentation Strategies

In this study, the efficacy of bioremediation strategies (enhanced natural attenuation with nitrate and phosphate addition [ENA] and bioaugmentation) for the remediation of creosote-contaminated soil (7767 ± 1286 mg kg^?1 of the 16 EPA priority PAHs) was investigated at pilot scale. Bioaugmentation of creosote-contaminated soil with freshly grown or freeze dried Mycobacterium sp. strain 1B (a PAH degrading microorganism) was applied following bench scale studies that indicated that the indigenous soil microflora had a limited PAH metabolic activity. After 182 days, the total PAH concentration in creosote-contaminated soil was reduced from 7767 ± 1286 mg kg^?1 to 5579 ± 321 mg kg^?1, 2250 ± 71 mg kg^?1, 2050 ± 354 mg kg^?1 and 1950 ± 70 mg kg^?1 in natural attenuation (no additions) and ENA biopiles and biopiles augmented with freshly grown or freeze dried Mycobacterium sp. strain 1B respectively. In ENA and bioaugmentation biopiles, between 82% and 99% of three-ring compounds (acenaphthene, anthracene, fluorene, phenanthrene) were removed while four-ring PAH removal ranged from 33 to 81%. However, the extent of PAH degradation did not vary significantly between the ENA treatment and biopiles augmented with Mycobacterium sp. strain 1B. Four-ring PAH removal followed the order fluoranthene > pyrene > benz[a]anthracene > chrysene. The high residual concentration of some four-ring PAHs may be attributable to bioavailability issues rather than a lack of microbial catabolic activity. Comparable results between ENA and bioaugmentation at pilot scale were surprising given the limited degradative capacity of the microbial consortia enriched from the creosote-contaminated soil.     

Large Scale Production of Erythritol and Its Conversion to d-Mannitol Production by n-Alkane-grown Candida zeylanoides

Production of erythritol by n-alkane-grown Candida zeylanoides KY6166 was studied using large scale fermentors under conditions described in the previous report. The medium-pH was kept below 4.0 during fermentation in 5-liter or 300-liter fermentors. This strain produced about 180mg/ml of meso-erythritol and a small amount of mannitol. The yield corresponded to 90% of n-alkane consumed.

The production ratio between erythritol and mannitol was affected remarkably by the concentration of phosphate in the medium. Keeping its concentration at high level (0.04 to 0.20 mg/ml as KH₂PO₄), erythritol production was almost entirely converted to mannitol production. The yield was 63 mg/ml after 100 hr incubation in 5-liter fermentor, which corresponded to 52% of n-alkane consumed. Conversion of the production of erythritol to mannitol was also observed in several other yeasts capable of utilizing n-alkane as the sole source of carbon.     

Development and Immunogenicity of a Brazilian Glycoconjugate vaccine against Meningococcal W in a Pilot Scale

Glycoconjugate Journal - Recent changes in the epidemiology of meningococcal have been reported and meningococcal group W (MenW) has become the third most prevalent group isolated in Brazil in the...     

微生物胞内酶(细胞)在气升式生物反应器中的大规模生产

气升式生物反应器由于周期变压和供氧特性在生产微生物胞内酶（细胞）时具有优良的性能,酶活力和稳定性的提高、发酵周期的缩短、单位能耗的降低、生产能力的提高,均显示了该反应器在传质、混合、流场特性、能耗等综合性能优越于机械搅拌发酵罐。     

亚硫酸盐废液中戊糖己糖同步酒精发酵中间试验

木材在亚硫酸盐法制浆过程中,占木材干重２０％～３０％的半纤维素降解成单糖而溶于废液中,废液中单糖的利用,不仅能够充分利用生物质资源,而且对于后续产品木质素的深加工十分必要。废液中糖主要由己糖和戊糖组成,近年来,随着阔叶材在制浆原料中的比例不断增加,废...     

红枫杜鹃的组培快繁与规模化生产

1植物名称 红枫杜鹃（Rhododenron molle×Rhododendron schlippenbachii）。 2材料类别 茎段、茎尖。 3培养条件 基本培养基为MS。（1）诱导愈伤组织培养基：1／4MS＋ZT4．0mg·L^-1（单位下同）＋NAA0．1＋2,4．D0．03;（2）继代培养基：1／4MS＋ZT1．0＋NAA0．1;（3）壮苗培养基：1／4MS＋ZT0．5＋NAA0．05;     

黑曲霉固态发酵木聚糖酶中试条件的研究

通过对黑曲霉在曲盘中进行固态发酵木聚糖酶中试条件进行优化研究,结果表明最适的产酶培养基中麸皮与玉米芯比例为6:4,添加硫酸铵可以促进黑曲霉菌丝生长,对酶活没有显著影响;最适的培养基初始pH值为4.0,培养基厚度为4cm;在28℃下培养60～84h,木聚糖酶活稳定在最高峰。     

人铜锌超氧化物歧化酶的大规模生产工艺

本文以干扰素生产中废弃的人红细胞为原料,采用乙醇－氯份沉淀→磷酸氢二钾盐析→丙酮沉淀手续和ＤＥＡＥ—５２层析步骤,进行了人铜锌超氧化物歧化酶（ＣｕＺｎ－ＳＯＤ）大规模生产工艺的实验研究。结果表明,生产的酶制剂中含量为９０．６％,比活性在２０００ＭｃＣｏｒｄ—Ｆｒｉｄｏｖｉｃｈ单位／ｍｇ蛋白以上,无菌、无热原质、安全性等项指标符合国家卫生部对血液制品的要求。     

Production of reactive nitrogen and oxygen intermediates in human granulocytes and monocytes during internalization of live BCG bacilli

The production of nitric oxide (NO) and reactive oxygen intermediates in granulocytes and macrophages from healthy volunteers, infected in vitro with live Bacille Calmette-Guerin (BCG) mycobacteria, was estimated. Significant differences in the biochemical reactions induced by BCG bacilli in granulocytes and monocytes are described. The activity of phagocytes was also investigated in the cultures with cytokines: IFN-gamma, TNF-alpha, GM-CSF, IL-4.     

Culture of Gigartina Skottsbergii (Rhodophyta) in Southern Chile. A Pilot Scale Approach

In the last 10 years studies on the management and exploitation of Chilean carrageenophytes have proliferated in response to the increasing development of the local processing industry. One of the most important sources of raw material for Chilean carrageenan, Gigartina skottsbergii Setchell et Gardner, was the subject of an intensive study to design a commercial cultivation technique which could be an alternative to wild harvest. In this context this pilot study reports the first successful attempt to culture G. skottsbergii from spores to harvestable plants. A three-step farming approach was developed: (i) seeding of spores onto scallop shells followed by a two-month nursery period in a greenhouse (until the development of initial upright thalli from the discoid crust occurred), (ii) outplanting juvenile plants on shells in the sea on a long-line system (until thalli attained 3–4 cm diameter) and (iii) detachment of fronds from the shells, fixing of individuals to vertical ropes and growth until commercial size was reached. Additional experiments to compare bottom and suspended growth, cultivation by fragmentation and whole fronds and meristematic activity of different zones of the fronds were performed. This study shows the technical feasibility of culturing G. skottsbergii from spores, complemented with growth of vegetative fragments, in order to optimize the management of the culture. In the future, therefore, it may be possible to replace the heavy exploitation of wild beds in southern Chile with farming activities.     

固定化生长酵母连续生产酒精的研究中间试验报告

造粒器与柱式生物反应器成—封闭系统,采用正交试验确定的最适固定化条件,以海藻酸钙凝胶法进行细胞固定化,连续造粒,在反应器中完成固定化。固定化酵母在反应器中通气培养20小时左右,凝胶球细胞数达2×10~9/me,其增长速度比传统工艺自然细胞快10倍。固定化生长细胞用于生物反应器连续发酵甜菜糖蜜酒精,酒精生产能力为22.1—23.67g/L凝胶球/小时,为传统工艺酒精生产能力的10倍。停留时间为3小时。反应器系统由两个0.7KL柱式反应器和1个0.8KL成熟醪接收器组成。发酵周期由传统工艺的20小时左右,缩短为4小时,酒精含量为8.5—9.0％(V/V),对1.2号反应器的动态变化及其在发酵中的作用进行了系统研究,糖蜜中可发酵糖75％的转化是在1号反应器中完成的。     

Stabilization of Enzymes against Thermal Stress and Freeze-Drying by Mannosylglycerate

2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications.